Post-transcriptional regulation of messenger RNA (mRNA) stability and translation is an important mechanism for rapidly controlling gene expression in response to stimuli, including environmental changes. This project seeks to generate and utilize structural information to enhance our understanding of these processes. PUF proteins are sequence-specific RNA-binding proteins that are important regulators of gene expression for embryonic development and germline stem cell maintenance. RNA silencing, the destruction of mRNA by double-stranded RNA containing corresponding sequences, is a powerful tool to knock out expression of target genes in eukaryotic cells and has therapeutic potential. In the past few years, much has been learned about the mechanism by which RNA silencing occurs, including the identification of proteins involved in the process. This project has two major focus areas. The first is to study the ability of PUF family proteins to regulate specific target mRNAs. Beginning with determining the first crystal structure of a PUF protein in complex with RNA to publishing work this year on the specificity of human and C. elegans proteins (refs. 1, 2), we have identified both common and unique features of RNA recognition by this family of proteins. The combination of the features in any particular protein results in a unique network of mRNAs that are regulated by that protein. Our studies on the RNA recognition properties of PUF proteins have allowed us to create artificial splicing factors in collaboration with Dr. Zefeng Wang's lab at the Univ. of North Carolina. Together we have demonstrated the ability to design factors that can regulate alternative splicing of endogenous pre-mRNA in cells. We have advanced this work by determining a recognition code for cytosine bases, which was not previously known (ref. 3). This new information expands our ability to recognize 8mer RNA sequences from 9,000 different sequences to more than 65,000. The second focus is to study proteins in the RNA silencing pathway. We use structural and biochemical methods to understand their functions. We have been investigating the substrate specificity and enzymatic mechanism of Dicer enzymes, which produce small interfering RNAs and microRNAs (ref. 4). Our work in collaboration with Dr. Phillip Zamores lab at the Univ. of Massachusetts Medical School demonstrates the roles of partner proteins, small molecule phosphate, and Dicers own N-terminal helicase domain in selecting substrates and processing them accurately. This RNA cleavage fidelity is crucial, because a shift of the cleavage site by just one base can alter the repertoire of mRNAs that are regulated.